X44 - Janus Base Nanopieces for mRNA Therapeutics with Engineered Endosomal Escape and Low Toxicity for Osteoarthritis Therapy

Introduction: : Over 32.5 million adults in the United States suffer from osteoarthritis (OA), a degenerative joint disease characterized by pain and discomfort due to the progressive deterioration of articular cartilage. The recent rise of mRNA therapeutics offers new hope for more effective OA treatment compared to protein-based drugs. However, for mRNA therapies to be successful in OA, the delivered mRNA must overcome endosomal entrapment within target cells while avoiding severe inflammatory responses that could further aggravate pre-existing OA-associated inflammation. In addition, effective delivery remains challenging due to the need to penetrate the dense extracellular matrix (ECM) such as cartilage. Here, we have specially designed and synthesized a new generation of Janus base nanotubes (self-assembled nanotubes inspired by DNA base pairs) to combine with mRNA to form rod-shaped delivery vehicles called Janus base nanopieces (JBNps). We designed this new generation of JBNps with a pore-forming property, enabling efficient early endosomal escape (in contrast to the late endosomal escape of most other delivery vehicles) while avoiding immune activation due to reduced membrane damage during endosomal escape—unlike lipid nanoparticles (LNPs), which are known to induce severe inflammation. Furthermore, the nano-rod morphology of JBNps offers a significant advantage in penetrating the dense extracellular matrix of articular cartilage. As proof of concept, we demonstrated that JBNps enable efficient delivery of IL1RA mRNA for the treatment of osteoarthritis.

Materials and

Methods: : The delivery of JBNp-mRNA was demonstrated by transfecting human chondrocytes (C28/I2) with eGFP-mRNA. Endosomal escape was assessed by staining cells with Galectin-9 and Alix antibodies and quantifying the puncta. In vivo delivery was evaluated by administering JBNp-mRNA-Cy5 to the mouse knee joint. To treat osteoarthritis (OA), mice with OA induced via Destabilization of the Medial Meniscus (DMM) surgery received weekly intra-articular injections of JBNp-IL1RA mRNA. Following treatment, extracted knee joints were analyzed by micro-CT imaging and stained with Safranin O/Fast Green. Behavioral assessments were performed, including thermal sensitivity (hot plate test), mechanical sensitivity (von Frey test), and gait analysis.

Results, Conclusions, and Discussions:: We designed and developed a nano-rod shaped delivery vehicle with a diameter of 20 - 30 nm (JBNps) capable of delivery to the intra-articular environment shown in TEM (Figure 1A). And demonstrated JBNp can effectively delivery eGFP-mRNA into human chondrocytes (Figure 1B). Pore-forming endosomal escape of JBNps enabled efficient escape from early endosomes, as indicated by Rab5 staining in a time-dependent manner (Figure 1C). Moreover, small pores formed during JBNp-mediated endosomal escape did not recruit Galectin-9, which senses large endosomal disruptions, whereas ALIX, a marker of endosomal repair, was preferentially recruited (Figure 1D). Furthermore, JBNp-mRNA-Cy5 was successfully delivered to the knee joints of DMM mice after eight weeks (Figure 1E). We obtained significant results such as improved gait measures including measurements of paw area, stance length, stride length, and sway length, fewer osteophytes shown in micro-CT, less cartilage degeneration (Figure 1F and 1G).

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